Human tumor related gene in region of sub-band 3 of band 3 of zone 1 of human chromosome 17 and detection kit

ABSTRACT

The invention provides a method for diagnosing susceptibility of individual cancer (e.g. liver cancer), comprising the steps of detecting individual HC56 gene, transcript and/or protein, and comparing it with normal HC56 gene, transcript and/or protein. The difference show the possibility of individual cancer is higher than that in the normal population. The invention also provides the corresponding detection kit.

FIELD OF INVENTION

This invention relates to the human tumor related gene HC56 in theregion of sub-band 3 of band 3 of zone 1 of human chromosome 17(17p13.3) and its uses. In particular, it relates to the method and kitfor detecting tumor susceptibility based on the single nucleotidepolymorphism or SNP of HC56.

PRIOR ART

The mortality rate of malignant tumor is just lower than that ofcardio-and cerebro-vascular disease in China. The mortality rate ofhepatocarcinoma is the third in common tumors, just below lung cancerand gastric cancer. The diagnostic and therapeutic means are lacking forhepatocarcinoma, except for microhepatocarcinoma. The development ofcarcinoma is a complex process involving multiple genes and steps andeffected by the activation of many oncogenes and inactivation ofanti-oncogenes. The anti-oncogenes are much important. Therefore, tofind anti-oncogenes is one of the focuses of the current studies. Theloss of heterozygosity (LOH) of a gene may result in the loss of normalcharacters. Anti-oncogenes may exist in the site having high frequencyof LOH.

The key state laboratory in Shanghai Cancer Institute has studied humanhepatocarcinoma gene for long time. In 1991, it was found that thedevelopment of hepatocarcinoma was related to the inactivation of p53,which had codon mutations not only on position 249 but also on otherpositions. (Li D. Z; Gu JR et al. Carcinogenesis 14 (2): 169, 1993). P53is located in chromosome 17p13.1.

The researches on 17p13.3 have been reported overseas. Nishida et al.selected 10 polymorphism probes on 17p and studied the relationshipbetween the LOH on 17p of human hepatocarcinoma and p53 mutation (NoashiNishida et al., Cancer Research 53: 368-372, 1993). He deemed that thedevelopment of hepatocarcinoma might be related to unknownanti-oncogenes on 17p13.3. Schultz et al. (David C. Schultz, et al.,Cancer Research 56: 1997-2002, 1996) proved that two new genes, OVCA1and OVCA2, were located on 17p13.3 by allele deletion mapping andsite-directed cloning. These two genes were expressed in normalepithelial cells of ovary, but were low-expressed or not expressed inoophoroma or ovary cancer cell lines. Wales et al. (Michele M. Wales etal., Nature Medicine: 570-577, 1995) discovered a candidate gene Hic-1on 17p13.3 by analyzing the DNA methylation degree in tumor tissues,which was a transcription factor having a zinc-finger structure and wasexpressed widely in normal tissues but low-expressed in tumor cells.

However, the previous reports did not indicate the minimum range of LOHin hepatocarcinoma tissue and the corresponding sites. Further, therelationship between the above candidate genes and hepatocarcinoma wasnot reported.

Since cancer is one of the main diseases harmful to human health, peopleare concerned about the early diagnosis and gene therapy of cancer so asto effectively cure and prevent tumors, such as hepatocarcinoma.Therefore, there is a keen need in the art to develop new tumor-relatedand/or tumor-inhibiting human proteins and the corresponding detectionkits.

SUMMARY OF INVENTION

One purpose of the invention is to provide a novel hepatoma relatedprotein, named HC56 protein, and its fragments, analogs and derivatives.

Another purpose of the invention is to provide a polynucleotide encodingthe polypeptides.

Still another purpose of the invention is to provide a method forHC56-based method and detection kit for detecting tumor susceptibility.

In the first aspect, the invention provides an isolated normal humanHC56 polypeptide, which comprises the amino acid sequence of SEQ ID NO:4. Further, it provides the amino acid mutation caused by SNPs whencompared with the normal human HC56. One example of the mutated HC56amino acid sequence is shown in SEQ ID NO: 2.

In the second aspect, the invention provides an isolated polynucleotideencoding the normal HC56 polypeptide, e.g., SEQ ID NO: 3. It furtherprovides the SNPs in human HC56 gene. One HC56 sequence having SNPs isshown in SEQ ID NO: 1.

In the third aspect, the invention provides a vector comprising theabove polynucleotide and a host cell transformed with the vector or ahost cell transformed with the polynucleotide.

In the fourth aspect, the invention provides a method for producing apolypeptide having the activity of HC56 protein, which comprises (a)culturing the above transformed host cell under the conditions suitablefor the expression of protein; (b) isolating the polypeptides having theactivity of HC56 protein from the culture.

In the fifth aspect, the invention provides nucleic acid molecules fordetection, which consist of 20-4750 consecutive nucleotides of SEQ IDNO: 3 and having mutations selected from the group consisting of:

-   -   SEQ ID NO: 3, position 2004, C→T;    -   SEQ ID NO: 3, position 2080, A→G;    -   SEQ ID NO: 3, position 2655, G→C;    -   SEQ ID NO: 3, position 3043, G→C;    -   SEQ ID NO: 3, position 3085, A→T;    -   SEQ ID NO: 3, position 3358, G→A;    -   SEQ ID NO: 3, position 4092, A→G;    -   SEQ ID NO: 3, position 4404, T→C;    -   SEQ ID NO: 3, position 4564, C→G;    -   SEQ ID NO: 3, position 4602, G→A.

Alternatively, the nucleic acid molecule has sequence of SEQ ID NO: 1.These nucleic acid molecules can be used as probes, primers or antisensefragments.

In the sixth aspect, the invention provides a method for detecting thecarcinomatous change or cancer susceptibility of hepatocytes, comprisingthe steps of: detecting whether there is any change of HC56 gene,transcript and/or protein in the hepatocyte of the subject when comparedwith the normal HC56 gene, transcript and/or protein, and the changeindicating that the possibility of suffering cancer in the subject ishigher than that in the normal population. Preferably, the change isnucleotide deletion, insertion or substitution.

In one preferred embodiment, the HC56 gene or transcript are detectedand compared with the normal HC56 nucleotide sequence and the change isselected from the group consisting of:

-   -   SEQ ID NO: 3, position 2004, C→T;    -   SEQ ID NO: 3, position 2080, A→G;    -   SEQ ID NO: 3, position 2655, G→C;    -   SEQ ID NO: 3, position 3043, G→C;    -   SEQ ID NO: 3, position 3085, A→T;    -   SEQ ID NO: 3, position 3358, G→A;    -   SEQ ID NO: 3, position 4092, A→G;    -   SEQ ID NO: 3, position 4404, T→C;    -   SEQ ID NO: 3, position 4564, C→G;    -   SEQ ID NO: 3, position 4602, G→A.

More preferably, the change is selected from the group consisting of:

-   -   SEQ ID NO: 3, position 3043, G→C;    -   SEQ ID NO: 3, position 3085, A→T;    -   SEQ ID NO: 3, position 4404, T→C;    -   SEQ ID NO: 3, position 4602, G→A.

In one preferred embodiment, the HC56 protein is detected and comparedwith the normal HC56 amino acid sequence and the change is selected fromthe group consisting of:

-   -   SEQ ID NO: 4, position 222, Pro→Ser;    -   SEQ ID NO: 4, position 247, Gln→Arg;    -   SEQ ID NO: 4, position 439, Glu→Gln;    -   SEQ ID NO: 4, position 568, Gly→Ala;    -   SEQ ID NO: 4, position 582, Glu→Val;    -   SEQ ID NO: 4, position 673, Arg→Gln;    -   SEQ ID NO: 4, position 926, Asn→Asp;    -   SEQ ID NO: 4, position 1022, Gys→Arg.

More preferably, the change is selected from the group consisting of:

-   -   SEQ ID NO: 4, position 568, Gly→Ala;    -   SEQ ID NO: 4, position 582, Glu→Val;    -   SEQ ID NO: 4, position 1022, Gys→Arg.

In the seventh aspect, the invention provides a kit for detecting tumor(e.g., hepatocarcinoma) susceptibility, which comprises primers whichspecifically amplify human HC56 gene or transcript to produce anamplification product having a SNP selected from the group consistingof:

-   -   SEQ ID NO: 3, position 2004, C→T;    -   SEQ ID NO: 3, position 2080, A→G;    -   SEQ ID NO: 3, position 2655, G→C;    -   SEQ ID NO: 3, position 3043, G→C;    -   SEQ ID NO: 3, position 3085, A→T;    -   SEQ ID NO: 3, position 3358, G→A;    -   SEQ ID NO: 3, position 4092, A→G;    -   SEQ ID NO: 3, position 4404, T→C;    -   SEQ ID NO: 3, position 4564, C→G;    -   SEQ ID NO: 3, position 4602, G→A.

Preferably, the kit further comprises the probes specifically binding tothe SNP site and/or a restriction enzyme specifically recognizing theSNP site wherein the nucleotide change in the SNP site results in theappearance or disappearance of the cleavage site of restriction enzyme.More preferably, the restriction enzyme is selected from SecI and EarI.

Preferably, the change is selected from the group consisting of:

-   -   SEQ ID NO: 3, position 3043, G→C;    -   SEQ ID NO: 3, position 3085, A→T;    -   SEQ ID NO: 3, position 4404, T→C;    -   SEQ ID NO: 3, position 4602, G→A.

The other aspects of the invention will be apparent to the skilled inthe art in light of the technical disclosure of the invention.

DESCRIPTION OF DRAWINGS

FIG. 1 and FIG. 2 show the expression of HC56 in hepatocarcinoma andsurrounding noncancerous tissues (RT-PCR), wherein G2, G4, G11, G12,G15, G16, D1, D22, D24 were from hepatocarcinoma patients. L:noncancerous liver tissues; K: cancerous liver tissues; +: positivecontrol; −: negative control; β2-M: β-microglobulin control probe.

FIG. 3 shows the expression of HC56 in normal human tissues. Lanes 1-8were heart, liver, brain, skeletal muscle, placenta, kidney, lung andpancreas, respectively.

FIG. 4 shows the Southern blotting of HC56 wherein the genomic DNA wasdigested by MSPI. G7, Q7, D40, G9, D2, D43 were from hepatocarcinomapatients. L: noncancerous liver tissues; K: cancerous liver tissues.

FIG. 5 shows the Southern blotting of HC56 wherein the genomic DNA wasdigested with EcoRI. D12, D13, D14 were from hepatocarcinoma patients.L: noncancerous liver tissues; K: cancerous liver tissues.

FIG. 6 shows the Southern blotting of HC56 wherein the genomic DNA wasdigested with EcoRI. G5, Q9, D10 were from hepatocarcinoma patients. L:noncancerous liver tissues; K: cancerous liver tissues.

FIG. 7 shows cell line 7721 transfected by vector PBK-CMV.

FIG. 8 shows cell line 7721 transfected by vector PBK-CMV-HC56.

FIG. 9 shows the construction scheme of expression vector pBR-CMV-CHx.

DETAILED DESCRIPTION OF INVENTION

In the hepatocarcinoma study, the inventors first found that there washigh frequency of LOH (60-100%) of 17p13.3 in hepatocarcinoma (HC)tissue. Recently, the full genomic scanning of HC also proved that17p13.3 was the region having the highest frequency. The inventorsisolated and cloned the tumor related ESTs or expressed sequence tags inregion 17p13.3 by screening the normal liver cDNA library using PAC P579clone corresponding to site 926 in region 17p13.3. The cDNA clone wasobtained. The full-length nucleotide sequence, named HC56, and theencoded amino acid sequence were obtained by RACE. The Northernblotting, RT-PCR and Southern blotting results proved that HC56 wasrelated to tumor. The in vitro experiment proved that HC56 inhibited thegrowth of liver cancer cell line 7721. The study also indicated thatthere were several SNPs in the exon regions of HC56. Therefore, HC56gene can be used in the diagnosis, treatment and prevention of tumor.

HC56 protein has various uses including, but not to be limited to:curing disorders (e.g., tumor) caused by low or no activity of HC56protein, and screening out antibodies, polypeptides or ligands asagonists of HC56. The expressed recombinant HC56 protein can be used toscreen polypeptide library to find out therapeutically valuablepolypeptide molecules that activate HC56 protein.

In another aspect, the invention also provides the polyclonal andmonoclonal antibodies, especially the monoclonal antibody, which arespecifically against the polypeptide encoded by human HC56 DNA orfragments thereof. By “specificity”, it is meant an antibody that bindsto the HC56 gene products or fragments thereof. Preferably, the antibodyspecifically binds to the protein having an amino acid sequence of SEQID NO: 2 and does not bind to the protein having an amino acid sequenceof SEQ ID NO: 4.

The present invention includes not only intact monoclonal or polyclonalantibodies, but also immunologically-active antibody fragments, e.g., aFab′ or (Fab)₂ fragment, an antibody light chain, an antibody heavychain, a genetically engineered single chain Fv molecule, or a chimericantibody.

The antibodies in the present invention can be prepared by varioustechniques known to those skilled in the art. For example, purified HC56gene products, or its antigenic fragments can be administrated toanimals to induce the production of polyclonal antibodies. Similarly,cells expressing HC56 or its antigenic fragments can be used to immunizeanimals to produce antibodies. Antibodies of the invention can bemonoclonal antibodies that can be prepared by using hybridoma technique.The polyclonal antibodies can be prepared by immunizing animals, such asrabbit, mouse, and rat, with HC56 protein. Various adjuvants, e.g.,Freund's adjuvant, can be used to enhance the immunization. The antibodyagainst HC56 protein can be used in immunohistochemical method to detectthe presence of HC56 protein in the biopsy specimen.

The invention further provides diagnostic assays for quantitative and insitu measurement of HC56 protein level. These assays are well known inthe art and include FISH assay and radioimmunoassay. The level of HC56protein detected in the assay can be used to illustrate the importanceof HC56 protein in diseases and to determine the diseases associatedwith HC56 protein.

A method of detecting the presence of HC56 protein in a sample byutilizing the antibody specifically against HC56 protein comprises thesteps of: contacting the sample with the antibody specifically againstHC56 protein; observing the formation of antibody complex, whichindicates the presence of HC56 protein in a sample.

The polynucleotide encoding HC56 protein can be used in the diagnosis ofHC56 protein related diseases. The polynucleotide encoding HC56 can beused to detect whether HC56 is expressed or not, and whether theexpression of HC56 is normal or abnormal, e.g., in the case of diseases.HC56 DNA sequences can be used in the hybridization with biopsy samplesto determine the expression of HC56. The hybridization methods includeSouthern blotting, Northern blotting and in situ blotting, etc., whichare public and sophisticated techniques. The corresponding kits arecommercially available. A part of or all of the polynucleotides of theinvention can be used as probe and fixed on a microarray or DNA chip foranalysis the differential expression of genes in tissues and for thediagnosis of genes. The HC56 specific primers can be used inRNA-polymerase chain reaction and in vitro amplification to detect thetranscripts of HC56.

Further, detection of the mutation of HC56 gene is useful for thediagnosis of HC56 protein related diseases. The mutation forms of HC56include site mutation, translocation, deletion, rearrangement and anyother mutations compared with the wild-type HC56 DNA sequence. Theconventional methods, such as Southern blotting, DNA sequencing, PCR andin situ blotting, can be used to detect mutation. A preferred method fordetecting the mutation of HC56 nucleic acid is to detect the presence ofSNPs. Moreover, mutation sometimes affects the expression of protein.Therefore, Northern blotting and Western blotting can be used toindirectly determine whether the gene is mutated or not.

The invention also provides a kit for detecting the presence of SNPs ofHC56 in the analyte, which comprises a suitable container, primerslocated in the container which specifically amplify HC56 gene ortranscripts to produce an amplification products having the SNP sitesidentified in the invention, and the specification.

The invention has the following characterizations: it has been provedthat the LOH or DNA rearrangement of HC56 exists in HC tissues bySouthern blotting; it has been proved that the expression of HC56 isinhibited in certain HC tissues by Northern blotting. Therefore, it issuggested HC56 is related to the development of HC and is a candidatefor HC anti-oncogene. The in vitro DNA transfection has shown that HC56remarkably inhibits the growth of HC cells. The SNP detection has shownthat at least two SNPs have sufficient statistical difference (p<0.01)between the HC patient peripheral blood DNA and normal peripheral bloodDNA. Therefore, the SNP detection kit is useful for detecting HCsusceptibility.

The invention is further illustrated by the following examples. It isappreciated that these examples are only intended to illustrate theinvention, but not to limit the scope of the invention. For theexperimental methods in the following examples, they are performed underroutine conditions, e.g., those described by Sambrook. et al., inMolecule Clone: A Laboratory Manual, N.Y.: Cold Spring Harbor LaboratoryPress, 1989, or as instructed by the manufacturers, unless otherwisespecified.

EXAMPLE 1 cDNA Cloning of HC56

DNA was extracted from P579 (Genome System Co.) and cut with NotI. TheDNA fragments about 100 bp were recovered and used as probes. Afterlabeling with ³²P-dATP, the probes were used to screen the human livercDNA library conventionally constructed. After pre-hybridization andhybridization, the positive clones were obtained.

The sequencing of one terminal sequence (about 500 bp) of cDNA cloneswas carried out on ABI 337 DNA auto-sequencer using dideoxy chaintermination method. After analysis, the sequencing of the other terminalwas carried out for some clones, which were determined to be new genes,until the full-length sequences were obtained. One clone was named HC56,whose nucleotide and amino acid sequences were shown in SEQ ID NOs: 3and 4. The full-length sequence was 4750 bp and the ORF was fromposition 1341 to 4484, encoding a protein having 1047 amino acids.

The homology comparison did not find any polypeptide or proteinhomologous to HC56.

EXAMPLE 2 Full Length Gene Preparation Using RT-PCR

The normal human liver tissue was taken, and the total RNA was extractedusing Trizol™ agents (GIBCO. BRL) according to the specification, andmRNA was extracted using mRNA Purification Kit (Pharmacia). The reversetranscription was carried out at 42 degree using MMLV-RT-Superscript II(GIBCO BRL) to obtain liver cDNA. Based on SEQ ID NO:3, the HC56specific primers were synthesized. The protocol was 97° C., 3 min, 1cycle; 94° C. 30 sec, 60° C. 30 sec, 72° C. 1 min, 35 cycles; 72° C. 10min, 1 cycles. The amplification product containing complete ORFsequence was obtained. The sequencing confirmed the sequence of theproducts was in accord with sequence of Example 1.

EXAMPLE 3 Construction of HC56 Recombinant Expression Vector

DNA was extracted from HC56 clone prepared in Example 1. According tothe scheme shown in FIG. 9, the extracted DNA was digested with EcoRI,ligated with pBK-CMV (Stratagene) digested with EcoRI, and transformedinto susceptible cells of XLI-blue. The white clones were selected andplasmid DNA was prepared therefrom and confirmed by enzymatic digestion.The resultant recombinant expression vector pBK-CMV-HC56 was obtained.

EXAMPLE 4 Semi-Quantitative RT-PCR of HC56

1 μl of total tissue RNA was taken. In a 20 μl reaction system, theSuperscript II RT kit (GIBCO. BRL) was used to synthesize the firststrand of cDNA. Then, the following primers were used in PCR:

HC56 upstream primer: 5′-ctgcccacca ccatctgcca-3′ (SEQ ID NO: 5)

HC56 downstream primer: 5′-agcagcatgg ccaacagggg-3′ (SEQ ID NO: 6).

β2-M was used as internal control DNA. After reaction, 3 μl PCR productwas taken and run on 6% PAGE. The results were shown in FIGS. 1 and 2.Compared with β2-M, the expression bands of HC56 in L were much higherthan those in K in Cases G2, G12, D24, were higher than those in K inCase G15, and were not different in other cases. It indicated that theexpression of HC56 was inhibited in certain HC tissues.

EXAMPLE 5 Northern Blotting of HC56 Gene with Multi-Tissue Membrane

The multi-tissue Northern hybridization membrane (MTN) commerciallyavailable from Clontech was pre-hybridized at 42° C. for 3-4 hrs(Pre-hybridization solution contained 50% formamide, 0.05M Tris, 1% SDS,5× Denhardt's solution, 0.1% sodium pyrophosphate, 100 mg/ml denaturedsperm DNA), hybridized with HC56 probes, and shown by X-rayautoradiography.

The results were shown in FIG. 3, indicating HC56 was highly expressedin heart, placenta and skeletal muscle, low expressed in lung, andexpressed in other tissues.

EXAMPLE 6 Southern Blotting of HC56

10 μg DNA extracted from human HC tissues and surrounding noncanceroustissues was completely digested by EcoRI and prepared into Southernmembrane according to the method described in “Molecule Clone: ALaboratory Manual”. The membrane was conventionally pre-hybridized,hybridized, and washed.

As shown in FIG. 4, the positions of HC56 in the HC tissue (K) andnoncancerous tissue (L) were different in Cases G7, D2, and D43,indicating DNA rearrangement in HC tissues.

As shown in FIGS. 5 and 6, there was one band of HC56 in thenoncancerous tissues (L), but no band in the HC tissues (K) in Cases D13and Q9, indicating DNA deletion. TABLE 1 LOH or DNA rearrangement ofHC56 in HC tissues No difference between HC cDNA Sample LOH DNArearrangement and noncancerous clone (pairs) in HC in HC tissue HC56 152 (D₁₃, Q₉) 4 (G₇, D₂, D₄₃, G₆) 9*D12, G7 and so on were case numbers.

EXAMPLE 7 In Vitro Transforming HC56 cDNA into Hepatocarcinoma Cell

In this example, the liposome kit was used to in vitro transformhepatocarcinoma cell.

(1) Cell line: primary hepatocellular carcinoma cell line 7721

(2) DNA: DNA from expression plasmid pBK-CMV-HC56

(3) Liposome: LIPOFECT AMINE™ Reagent Kit (BRL Co.)

(4) Medium: non-serum medium SF-DMEM

-   -   Full-nutrient medium (supplemented with 10% FBS)    -   Full-nutrient medium containing G418    -   T25 culture flask.

(5) Preparation of DNA-liposome complex: 50 μl lipofectin was mixed with50 μl SF-DMEM. DNA (20 μg, in 20 μl TE) was added into 80 μl SF-DMEM,and mixed. The diluted DNA was added into diluted lipofectin solution,mixed for 5-10 mins at room temperature. 1.3 ml SF-DMEM was added intoDNA-lipofectin complex and the final volume was 1.5 ml.

(6) Transfecting cells: It was preferred that cells were grown to 80-90%confluence. Before experiment, the culture medium was changed once. 1.5ml lipofectin—DNA complex was added onto the cell surface, gently shakenand mixed homogeneously, incubated at 37° C. for 1-3 hrs. 1.5 ml SF-DMEMwas added and mixed homogeneously, and cells were incubated at 37° C.for 1-3 hrs. The culture medium was changed and cells were incubated at37° C. overnight. When the confluence of cell was 70% , the medium waschanged by the medium containing G418. Then the medium was changed asbefore until clones appeared.

The results were shown in FIGS. 7 and 8 and Table 2. In FIG. 7, the 7721cells transfected by blank expression vector (pBK-CMV) formed manyclones, while in FIG. 8, the 7721 cells transfected by expression vectorcontaining HC gene formed no or only few clones. It indicated that HC56in vitro inhibited the growth of HC cells. TABLE 2 In vitro inhibitionof HC56 on the growth of HC cells cDNA Blank vector Positive controlclone pBK-CMV p21 HC56 Clone numbers (mean) 11 0 0.3

EXAMPLE 8 SNPs Detection

The nested PCR primers and sequencing primers were constructed by usingABI3948 oligo synthesizer.

DNA was prepared conventionally from HC tissues, surroundingnoncancerous tissues, and peripheral blood from patients. Meanwhile, theDNA from normal peripheral blood was prepared as control. The reactionsystem having a total volume of 50 μl contained 50 ng genomic DNA. ThePCR was conducted in standard condition. The sequencing was carried onABI 377 plate gel using ABI big Dye Terminator Chemistry, or usingABI3700 capillary sequencer. The SNPs or mutations were collected bySequence Navigator™.

80 pairs of HC tissues and the surrounding noncancerous tissues wereanalyzed for SNPs in the exons of HC56 gene. There were 13 SNPs in theexons, 8 of which was accompanied by amino acid changes. Further, onceposition 3085 had a SNP, it was sure 3043, 4404, and 4602 also had SNPs.The results were shown in Table 3. TABLE 3 High Frequency of cSNPNucleotide Amino Nucleotide Amino acid no acid no alteration AlterationLinkage 2004 222 C→T Pro→Ser 2080 247 A→G Gln→Arg 2655 439 G→C Glu→Gln3043 568 G→C Gly→Ala + 3085 582 A→T Glu→Val ⊕ 3358 673 G→A Arg→Gln 4092926 A→G Asn→Asp 4404 1022 T→C Gys→Arg + 3′UTR 4564 C→G 4602 G→A +

70 patients' peripheral blood DNA and 100 normal persons' peripheralblood DNA were detected for SNPs on positions 3043 and 3085. It wasfound that the frequencies of SNPs in HC patients' peripheral blood DNAwere higher than those in the normal person and had sufficientstatistical difference (p<0.01), indicating HC56 was a susceptibilitygene for The results were shown in Table 4. TABLE 4 cSNP in C-56 and HCCNormal Hepatocellular Carcinoma PBC(a) PBC(b) Non-Cancerous HCC (%) (%)Liver(%) (%) SNP n = 100 n = 70 n = 80 n = 80 3043bp G→C (368aa)(Gly→Ala) G/G 70.0 41.4 (29) 42.5 (34) 45.0 (36) G/C 26.0 50.0 (35) 50.0(40) 42.5 (34) C/C 4.0 8.6 (6) 7.5 (6) 12.5 (10) (a Vs b p = 0.001)*3085bp A→T (582aa) (Glu→Val) A/A 95.0 80.0 (56) 76.3 (61) 78.7 (63) A/T5.0 18.6 (13) 22.5 (18) 18.8 (15) T/T 0.0 1.4 (1) 1.2 (1) 2.5 (2) (a Vsb p = 0.008) 4404bp T→C (1022aa) (Cys→Arg) T/T 57.0 40.0 (28) 45.0 (36)43.7 (35) T/C 36.0 51.4 (36) 45.0 (36) 41.3 (33) C/C 7.0 8.3 (6) 10.0(8) 15.0 (9) 4602bp G→A 3′UTR G/G 75.0 77.1 (54) 75.0 (60) 75.0 (60) G/A22.0 21.4 (15) 21.2 (17) 17.5 (14) A/A 3.0 1.42 (1) 3.8 (3) 7.5 (6)*3085 A→T alteration linked with 3043, 4404, 4602, polymorphism in liver& HCC.

EXAMPLE 9 Detection Kit

The hepatocarcinoma susceptibility detection kit was prepared, whichcomprised a pair of primers amplifying the SNPs on positions 3043 and3085.

Upstream primer: 5′-tgtggagggt tttcaggaag-3′ (SEQ ID NO: 7)(corresponding to positions 2861-2880 in SEQ ID NO: 1)

Downstream primer: 5′-tggagccagt attcctctcg-3′ (SEQ ID NO: 8)(corresponding to 3357-3376 in SEQ ID NO: 1).

Further, the kit comprised restriction enzymes SecI and EarI, which wereuseful for detect the SNPs on positions 3043 and 3085. For the SNP onposition 3043, once G was changed into C, the product could not bedigested with SecI while the unchanged amplification product could bedigested with SecI. For the SNP on position 3085, once A was changedinto T, the product could not be digested with EarI while the unchangedamplification product could be digested with EarI. Therefore, the kitcould easily detect the SNPs on positions 3043 and 3085 by digesting theamplification products.

All the documents cited herein are incorporated into the invention asreference, as if each of them is individually incorporated. Further, itwould be appreciated that, in the above teaching of the invention, theskilled in the art could make certain changes or modifications to theinvention, and these equivalents would still be within the scope of theinvention defined by the appended claims of the present application.

1. A method for detecting cancer susceptibility in a subject comprisingthe steps of: detecting whether there is any change of HC56 gene,transcript and/or protein in the subject when compared with the normalHC56 gene, transcript and/or protein, and the change indicating that thepossibility of suffering cancer in the subject is higher than that inthe normal population.
 2. The method of claim 1 wherein the HC56 gene ortranscript are detected and compared with the normal HC56 nucleotidesequence and the change is selected from the group consisting of: SEQ IDNO: 3, position 2004, C→T; SEQ ID NO: 3, position 2080, A→G; SEQ ID NO:3, position 2655, G→C; SEQ ID NO: 3, position 3043, G→C; SEQ ID NO: 3,position 3085, A→T; SEQ ID NO: 3, position 3358, G→A; SEQ ID NO: 3,position 4092, A→G; SEQ ID NO: 3, position 4404, T→C; SEQ ID NO: 3,position 4564, C→G; SEQ ID NO: 3, position 4602, G→A.
 3. The method ofclaim 2 wherein the change is selected from the group consisting of: SEQID NO: 3, position 3043, G→C; SEQ ID NO: 3, position 3085, A→T; SEQ IDNO: 3, position 4404, T→C; SEQ ID NO: 3, position 4602, G→A.
 4. Themethod of claim 1 wherein the HC56 protein is detected and compared withthe normal HC56 amino acid sequence and the change is selected from thegroup consisting of: SEQ ID NO: 4, position 222, Pro→Ser; SEQ ID NO: 4,position 247, Gln→Arg; SEQ ID NO: 4, position 439, Glu→Gln; SEQ ID NO:4, position 568, Gly→Ala; SEQ ID NO: 4, position 582, Glu→Val; SEQ IDNO: 4, position 673, Arg→Gln; SEQ ID NO: 4, position 926, Asn→Asp; SEQID NO: 4, position 1022, Gys→Arg.
 5. The method of claim 4 wherein thechange is selected from the group consisting of: SEQ ID NO: 4, position568, Gly→Ala; SEQ ID NO: 4, position 582, Glu→Val; SEQ ID NO: 4,position 1022, Gys→Arg.
 6. A kit for detecting tumor susceptibilitywhich comprises primers that specifically amplify human HC56 gene ortranscript to produce an amplification product having a SNP changeselected from the group consisting of: SEQ ID NO: 3, position 2004, C→T;SEQ ID NO: 3, position 2080, A→G; SEQ ID NO: 3, position 2655, G→C; SEQID NO: 3, position 3043, G→C; SEQ ID NO: 3, position 3085, A→T; SEQ IDNO: 3, position 3358, G→A; SEQ ID NO: 3, position 4092, A→G; SEQ ID NO:3, position 4404, T→C; SEQ ID NO: 3, position 4564, C→G; SEQ ID NO: 3,position 4602, G→A.
 7. The kit of claim 6 which further comprises theprobes specifically binding to the SNP site and/or a restriction enzymespecifically recognizing the SNP site wherein the nucleotide change inthe SNP site results in the appearance or disappearance of the cleavagesite of restriction enzyme.
 8. The kit of claim 7 wherein therestriction enzyme is selected from SecI and EarI.
 9. The kit of claim 6wherein the change is selected from the group consisting of: SEQ ID NO:3, position 3043, G→C; SEQ ID NO: 3, position 3085, A→T; SEQ ID NO: 3,position 4404, T→C; SEQ ID NO: 3, position 4602, G→A.
 10. A nucleic acidmolecule which consists of 20-4750 consecutive nucleotides of SEQ ID NO:3 and having mutations selected from the group consisting of: SEQ ID NO:3, position 2004, C→T; SEQ ID NO: 3, position 2080, A→G; SEQ ID NO: 3,position 2655, G→C; SEQ ID NO: 3, position 3043, G→C; SEQ ID NO: 3,position 3085, A→T; SEQ ID NO: 3, position 3358, G→A; SEQ ID NO: 3,position 4092, A→G; SEQ ID NO: 3, position 4404, T→C; SEQ ID NO: 3,position 4564, C→G; SEQ ID NO: 3, position 4602, G→A, or has thenucleotide sequence of SEQ ID NO: 1.